Resumen de Strategies for rapid and reagent-less electrochemical detection of rpa products

Sallam Mohammed Ahmed Al Madhagi

• Nowadays, there is a need to develop a rapid, simple, inexpensive and reliable DNA testing system for diagnosis in different fields such as genetic diseases, pathogens detection, forensics, and personalised medicine.

  In this work, the isothermal amplification and modified tailed primers to simplify the steps required for the electrochemical DNA detection are combined.

  Modified tailed primers are based on a single stranded oligonucleotide sequence linked to a carbon spacer, which effectively blocks elongation, prior to the primer sequence. Thus, resulting in an amplicon with a duplex flanked by two single stranded DNA tails. One of the tails was used to hybridise to a surface immobilised probe and the other to an enzyme or gold nanoparticles labelled reporter probe. Using these modified primers allowed us to detect DNA electrochemically without any need for post-amplification sample treatment decreasing the assay time and presenting an approach that can facilely find the application at the point of need.

  In this work, three different methods have been proposed and investigated in parallel based on using modified primers and isothermal amplification.

  In the first method (Chapters 2), an extremely rapid and sensitive DNA amplification and detection assay has been developed based on combining tailed primers and isothermal amplification. Using the HLA-DQB1*02 allele as a model system, the sensor demonstrated the ability to amplify and detect DNA in a few minutes without the need to perform any post- amplification treatments.

  In the second method (Chapters 3), a geno-sensor has been developed for the direct detection of the RPA products without the need to purify, create ssDNA or perform labelling process. The sensor developed based on using a forward primer modified with ssDNA tail and reverse primer-linked with HRP. Using this combination of primers allow us to produce an amplicon of duplex target specific DNA, flanked by single-stranded DNA tail on one end and HRP on the other using karlodinium armiger DNA as a model system.

  In the third method (Chapters 4), a reagent-less geno-sensor has been developed based on using tailed primers and AuNPs modified with thiolated ssDNA and 6-(Mercaptohexyl)ferrocene as an active redox label for electrochemical detection. Using these modified primers along with modified AnNPs allowed us to detect karlodinium armiger DNA electrochemically without any need for post-amplification sample treatment.